JP2001013136A - Histamine measuring method and histamine measuring device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a histamine measuring method and a histamine measuring device, capable of quantitating histamine directly by sing a histamine acceptor expression cell for detection of histamine. SOLUTION: A histamine acceptor expression cell is stimulated by histamine of plural known concentrations to obtain a concentration-response curve (calibration curve) between a current quantity of the cell and the histamine concentration. An antigen 12 is reacted with collected whole blood 11, and the histamine acceptor expression cell 2 is stimulated by the whole blood 11 after stimulus by the antigen, and the changing quantity of the current is applied to the calibration curve, and the histamine concentration included in the whole blood 11 after stimulus by the antigen is quantitated, to obtain a histamine liberation rate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention is intended to provide a method for quantifying histamine present in a sample, particularly in blood or cell culture. As a measurement system, a cell reaction of a histamine receptor-expressing oocyte is particularly used for detecting histamine.

[0002]

2. Description of the Related Art The histamine release test method is a method of stimulating leukocytes in blood and mast cells in mucosal tissues, releasing histamine from the cells, and quantifying the released histamine. This method has been reported as a method to identify allergens in allergic diseases (Kazuaki Tabe: Diagnosis of allergy-histamine release test, SRL Houka Vol.21, No.1-
2, 1997). Regarding the method for measuring free histamine, for example, histamine is separated by a fluorescent high performance liquid chromatography (HPLC) method as disclosed in JP-A-6-331619 and then reacted with a fluorescent reagent to measure the amount of fluorescence. A method of separating free histamine by a glass fiber as described in Kaihei 10-62415 or JP-A-10-170514, reacting it with a fluorescent reagent, and measuring the amount of fluorescence, furthermore, ICN Pharma
ELISA by competitive assay sold by ceuticals and Immunotech, and the like are known.

[0003]

In the conventional histamine release test method by measuring the amount of fluorescence, only histamine is separated from a sample, the separated histamine is labeled, and the amount of the labeled compound is defined as the amount of free histamine. The present invention provides a method for directly quantifying histamine by using a histamine receptor in contrast to this indirect histamine quantification method. In addition, by using a histamine receptor, a method for recognizing histamine with high specificity is provided even when a sample is contaminated with a histamine derivative or a histamine analog or a very small amount of histamine is contained. Things. It is another object of the present invention to provide a simple method for quantifying histamine without using complicated receptors such as purifying a sample by using a highly specific receptor. Further, the present invention provides a method for quantifying histamine in a shorter measurement time since histamine can be directly recognized and histamine labeling and purification are not required by using a highly specific receptor.

[0004] Further, a detection method is to directly convert a physiological response of a histamine receptor-expressing oocyte when the oocyte recognizes histamine into an electric signal. Cell reactions are known to occur in milliseconds or less, and histamine can be detected within 1 second. That is, the present invention provides a method capable of judging the presence or absence of histamine extremely quickly as compared with other detection methods.

[0005] Here, the functions and problems generally required for the histamine release test can be summarized as follows.

(1) Histamine, not a labeled compound, can be directly quantified.

(2) Measurement can be performed without pretreatment of the sample.

(3) The results can be obtained quickly.

(4) A highly specific measurement method.

Accordingly, an object of the present invention is to provide a histamine release test method for achieving the above-mentioned object.

[0011]

The histamine release test method of the present invention comprises adding whole blood after antigen stimulation to cells in which a histamine receptor has been previously expressed, and detecting the cell response. A certain amount of an antigen is added to the collected whole blood, and the mixture is reacted at 37 ° C. for about 10 to 30 minutes. The reaction time is only a guide and may be changed by optimizing an allergic reaction. At this time, it is desirable to shake lightly. During this time, the histamine receptor-expressing cells are stimulated with a plurality of known concentrations of histamine, and a concentration-response curve (calibration curve) between the reaction amount of the cells and the histamine concentration is determined. The histamine receptor-expressing cells are stimulated with whole blood after antigen stimulation, and the amount of the reaction is fitted to a calibration curve to determine the histamine concentration contained in whole blood after antigen stimulation. The histamine release rate is obtained by dividing the histamine concentration of whole blood after antigen stimulation by quantifying the histamine concentration contained in whole blood obtained by releasing histamine contained in blood cells by freezing and thawing whole blood. Ask by. Further, a buffer solution is added instead of the antigen, and the spontaneous free concentration of histamine from cells in the blood is determined using whole blood under the same conditions (control). By using this method, a histamine release test can be performed in about 20 to 50 minutes.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The histamine release test method of the present invention comprises adding whole blood after antigen stimulation to cells in which a histamine receptor has been expressed in advance, and detecting the cell response. In the following, a description will be given using an electrophysiological detection method of the response of the chloride ion channel through the histamine receptor as a cell reaction detection method, but is not necessarily limited to the chloride ion channel response and the electrophysiological detection method, As described in Japanese Patent Application Laid-Open No. 9-242377, other methods include cell responses derived from stimulation of receptors and methods for detecting the same.

An embodiment of the present invention will be described below with reference to the drawings.

FIG. 1 is a schematic diagram of a histamine quantification method using histamine receptor-expressing oocytes. The histamine receptor-expressing oocyte 2 is placed on the body part 1 filled with the buffer.
Two glass electrodes 3 and 4 connected to a silver wire coated with silver chloride coated with 3 M KCl are inserted into the oocyte 2. electrode
The readout from 3 is sent to the differential amplifier 6 and the recording device 7 as a potential difference from the external electrode 5, and is recorded. The differential amplifier applies a current of a difference between the signal sent from the electrode 3 to the differential amplifier 6 and the fixed potential to the oocyte through the electrode 4. Histamine 8 was added to the oocytes fixed in this way.
Response of the cell by dropping
Can be obtained.

FIG. 2 shows the relationship between the histamine concentration obtained by the above method and the cell response. The histamine concentration and the current change amount, that is, the cell current change amount, have a positive correlation in the range of the histamine concentration of 20 to 200 nM. That is, the concentration of histamine contained in the sample can be determined from the amount of change in the current of the cell.

FIG. 3 shows the current response of cells caused by histamine in blood. When histamine-containing blood is added to histamine receptor-expressing oocytes, there is a change in cell current as shown in A. On the other hand, when blood containing no histamine is added, no current change occurs as shown in B. In other words, the current change indicated by A is not caused by other contaminants in blood, but is caused in response to histamine. That is, in this method, histamine in blood is separated and
It can be quantified as it is without purification and labeling.

Example 1 FIG. 4 is a diagram showing a basic protocol when the present method is applied to allergy diagnosis. A certain amount of antigen 12 such as cedar pollen is added to the collected whole blood 11, for about 10-30 minutes, 37
React at ° C. Histamine 8 is released from basophils 13 in blood by antigen stimulation. During this period, the histamine receptor-expressing oocytes are stimulated with a plurality of known concentrations of histamine, and a concentration-response curve (calibration curve) between the amount of change in the current of the cells and the histamine concentration is obtained in the same manner as in FIG. Whole blood after antigen stimulation is collected with a pipette or the like to stimulate histamine receptor-expressing cells. The amount of electrical response is fitted to a calibration curve, and the concentration of histamine contained in whole blood after antigen stimulation is quantified. The histamine release rate is the histamine concentration of whole blood released by antigen stimulation, B is the histamine concentration of whole blood released from all histamine contained in blood cells by freeze-thawing method, and B is the control instead of antigen. And the concentration of spontaneously free histamine from the cells in the blood is determined as C by (AC) / B.

By using this method, Japanese Patent Application Laid-Open
The measurement time is as short as about 30 minutes as compared with 3 hours according to the method of 2415 or JP-A-10-170514 and 4 hours according to the ELISA method. Also, the number of work steps is four steps, which is extremely small compared to other methods. Another feature is that no reagents other than the standard histamine for directly measuring the histamine concentration are required, and the cost can be reduced.

Example 2 FIG. 5 shows the results of quantification of the concentration of histamine released from cells in the blood into plasma by stimulation with antigen (cedar pollen) in whole blood and using this method. The value obtained by subtracting the amount of histamine (control) released when stimulated with the buffer alone from the amount of free histamine was divided by the amount of histamine obtained by freezing and thawing whole blood to obtain the histamine release rate by antigen stimulation. I asked. When the histamine release rate by a specific antigen stimulation is 10% or more in a general histamine release test, the allergic reaction of the sample is considered to be positive. That is, it is considered that the specimens 1 and 3 have an allergic reaction to cedar pollen. These results were consistent with the conventional HRT test and the subject's subjective symptoms.

EXAMPLE 3 FIG. 6 shows an actual reaction vessel for performing an allergy test using whole blood. This container holds oocytes and uses a glass electrode
Measure the current change in steps 3 and 4; immerse the oocytes in a fixed volume of buffer solution; drain the water to wash the inside of the container; and transfer allergic blood to the oocytes. It consists of a part to be added.

Next, the actual measuring method will be described in detail. A buffer solution for oocytes is allowed to flow through the water channel 14 to fill the container, and then an excess liquid is discharged from the water channel 15 by a vacuum pump or the like. Thus, a constant amount of liquid sufficient to soak the oocyte can be always maintained. Oocytes 2 cultured 2-3 days after introduction of the histamine receptor mRNA are gently submerged in the solution,
It is fixed in the oocyte hole 16. The glass electrode 3 is inserted into the oocyte to check the membrane potential of the oocyte. Next, the glass electrode 4 is stabbed into the oocyte, and the membrane potential is fixed at -60 mV.

Thereafter, the whole blood 11 prepared by the operation shown in FIG. 4 is gently added to the oocyte 2 with a pipette 9. Whole blood 11
Contains histamine 8 released by histamine release reaction
It is included. When the histamine stimulates the oocyte 2, the current change can be measured. The amount of histamine in whole blood can be determined from the magnitude of the current change.

In addition to the method of adding the whole blood that has undergone the histamine release reaction with a pipette 9, a part for performing the histamine release reaction of whole blood is attached to a part of the container, and the histamine release reaction of whole blood It is also possible to continuously measure up to the quantification of the amount of histamine release accompanying the reaction.
A small amount of 5-20 mL of blood is directly injected into the glass capillary 17. The inner wall of the glass capillary 17 is already coated with the antigen 12 such as pollen. Heat the glass capillary 17 with the oocyte reaction vessel
Stab at 18. The heater section 18 is set at a predetermined temperature by a temperature control section (not shown). The glass capillary 17 is warmed at 37 ° C. for about 30 minutes to cause a histamine release reaction. In this embodiment, the glass capillary 17 is heated to 37 ° C., but may be in the range of 30 to 45 ° C. at which the histamine release reaction occurs. After that reaction, cock
19 is opened, the whole blood 11 inside the glass capillary 17 is added to the oocyte 2 by sending means (not shown), and the current response is measured.

After measuring a series of reactions of the oocyte, the glass electrodes 3 and 4 are extracted from the oocyte 2 and the oocyte 2 is discharged together with the solution from the drainage channel 20 by suction. Wash water flows from the water channel 15 and is discharged from the drain channel 20. Perform these operations 3-
Repeat 4 times to remove dirt from inside the container.

[0025]

According to the present invention, by using histamine receptor-expressing cells for histamine detection, histamine can be directly quantified, not as a labeling compound, so that the number of working steps and the number of reagents used can be minimized. Therefore, cost and measurement time can be reduced. Further, from these characteristics, free histamine in whole blood can be quantified in a short time without any pretreatment.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a histamine quantification method using histamine receptor-expressing oocytes.

FIG. 2 is a graph showing the relationship between histamine concentration and cell response (calibration curve).

FIG. 3 is a diagram showing a cellular response to histamine in blood.

FIG. 4 shows the histamine release test protocol.

FIG. 5 is a tabular diagram showing measured values of blood histamine concentration according to the present method.

FIG. 6 is a view showing a container for an allergy test.

[Explanation of symbols]

1 ... body part, 2 ... histamine receptor-expressing oocytes, 3.
..Glass electrode, 4 ... Glass electrode, 5 ... External electrode, 6 ... Differential amplifier, 7 ... Recording device, 8 ... Histamine, 9 ... Pipette, 10 ... Cell response, 11: Blood (whole blood), 12: Histamine-releasing substance such as antigen, 13: Basophil, 14: Buffer injection groove, 15: Water channel, 16 ... Hole for oocyte, 17 ・ ・ ・ Glass capillary, 18 ・ ・ ・ Heating part, 19 ・ ・ ・ cock, 20 ・ ・ ・
Drains.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/483 G01N 33/49 Z 33/49 33/53 Q 33/53 27/46 336G F term (reference) 2G045 AA01 BA13 BB14 BB22 BB46 BB51 DA71 FB03 GC20 JA07 4B029 AA07 BB11 CC03 FA12 FA15 4B063 QA01 QA19 QQ03 QQ61 QR77 QS39 QX04

Claims (11)

[Claims] 1. A method for measuring histamine comprising quantitatively measuring a histamine-dependent reaction of an oocyte having a histamine receptor, and quantifying histamine contained in a sample. 2. The method for measuring histamine according to claim 1, wherein unlabeled histamine is detected. 3. The method for measuring histamine according to claim 1, wherein unpurified histamine is detected from the contaminants. 4. The method for measuring histamine according to claim 1, wherein histamine contained in blood is detected and quantified by using a histamine receptor-expressing oocyte in the histamine detecting section. 5. A container having a hole for holding an oocyte, and a tube for removing a waste liquid from the container, wherein a histamine release reaction is caused by the blood to be measured and the antigen, A histamine measuring device comprising: adding a liquid to the container; measuring a change in the amount of current of the oocyte; and determining an amount of histamine released by the histamine release reaction. 6. The histamine measuring apparatus according to claim 5, wherein the reaction solution is added from above the container using a flow channel or a pipette. 7. The histamine measuring apparatus according to claim 5, further comprising a reaction tank for causing a histamine release reaction from histamine-containing cells such as blood, leukocytes, and mast cells. 8. The histamine measuring device according to claim 7, further comprising a liquid sending means for sending the reaction solution from the reaction tank to the container. 9. A container having a hole for holding an oocyte, a capillary in which an antigen is fixed on a wall surface, and a pump for sending a reaction solution in the capillary to the container in which an oocyte is held. A liquid means, and a tube for removing waste liquid from the container, causing the blood to be measured to flow through the capillary, causing a histamine release reaction in the capillary, and measuring a change in the amount of current of the oocyte. A histamine measuring device for determining an amount of histamine released in the capillary. 10. The histamine measuring apparatus according to claim 9, further comprising temperature control means for controlling said capillary at a predetermined temperature. 11. The predetermined temperature is 30 ° C. or more and 45 ° or more.
The histamine measurement device according to claim 10, wherein the histamine measurement device is not more than C.